Dot Plots Colored by Channel

In a dot plot colored by channel, each event in a typical biaxial plot is colored by its expression on a third channel, called the coloring channel. This functionality is available in the gating interface and the Working Illustration, with slightly different methods of use. Click the links below to jump to the relevant section.

Dot plots colored by channel have an important role in viSNE workflows. When looking at a viSNE map, coloring by channel allows one to correlate spatially collocated groups of events with biological populations:

(click to expand - the same sample with viSNE map colored deferentially by channel)

Dot plots colored by channel with signaling

Consider the below example of a simple signaling experiment. The coloring channel is pSTAT3. The two samples are unstim and IL10 stimulated PBMCs. It can be seen that general T cell signaling is happening in the sample stimulated with IL10.

(click to expand - dot plots colored by channel reveal signaling)

Dot plots colored by channel can help with gating

Coloring dot plots by channel can help draw gates in the right location, by displaying more information to interpret while drawing the gate. This can especially help in situations where drawing a clean gate is challenging.

Gating Interface - Dot Plots Colored by Channel

In the gating interface plot settings box, choose a plot type of dot plot colored by channel. Other important settings are indicated but discussed in other parts of the article.

(Gating Interface configuration for dot plots colored by channel)

The option for coloring channel appears under the gating plot beneath the X and Y axis controls. Each dot on the plot is colored according to its data value in the coloring channel.

Working Illustration - Dot Plots colored by Channel

In the Working Illustration, first choose a plot type of dot, then open the color by menu and choose the option for channel. After this, the selector for coloring channel will define what channel your plots are colored by. For figures that display many plots simultaneously with rotating coloring channels (see viSNE example above), select panel/channel values in the coloring channel menu and configure the Channels Figure Dimension box with the channels of interest for your figure. Configuration is indicated below. Every option seen in the image is important for dot plots colored by channel and discussed elsewhere in this article.

(Working Illustration configuration for dot plots colored by channel)

Dot Plotting Method - Binned versus Stacked

There are two plotting methods for dot plots colored by channel: Binned Pixels and Stacked Dots.

Binned Pixels

In Binned Pixels mode, collisions between events are binned into single pixels based on the chosen resolution of the plot. Each pixel then reports the median value of the events inside of it on the coloring channel selected for the plot.

Stacked Dots with Custom Size and Shape

The Stacked Dot option arranges events on top of each other depending on the order in which they appear in the FCS file. The size of each event in pixels can be adjusted with the Dot Size field. This functionality is useful for increasing visual quality of sparse data. No binning happens with stacked dots, so data can be hidden by overlap in some cases. The Dot Shape can be configured as well to be a circle, ring, or square. The ring shape is hollow in the middle and can help visualize events that might be overlapping because of stacking. Changing dot shape has little impact unless larger plot resolutions and dot sizes are used.

(click to expand - comparison of dot plotting method and shape. All plots are 512 pixel resolution. All stacked dot images are dot size of 7)

Coloring Channel Range

The Coloring Channel Range or Color Bar Range is the minimum and maximum of the color bar which is shown to the right of any dot plot colored by channel. The color bar is the key for associating a color in the dot plot to a particular value for expression intensity. The minimum and maximum values of the color bar are determined according to the selected method:

Global (all files): The ungated 2nd and 98th percentile values for the channel in question are calculated for all files in the experiment. The smallest 2nd and largest 98th percentile value are used as the coloring range minimum and maximum for all files in the experiment on this channel.

Local (per file): The ungated 2nd and 98th percentile values are calculated for the channel in question and assigned as coloring range minimum and maximum on a per-file basis.

User-defined: The coloring range boundaries are set through user-defined values. Currently only one custom range can be defined at a time and is not channel-specific.

The data values that define coloring behavior and range are calculated on data scaled according to the scaling method of the channel coloring the plot (and compensated, as applicable). However, the data values are untransformed for reporting in the color bar. The coloring follows a linear relationship through the transformed data, but not in the raw data. This is why for non-linearly transformed scales (arcsinh, log) the raw values reported in the color bar do not grow linearly.

Coloring Palette

The same color palettes available to other plot types in Cytobank are available for dot plots colored by channel. The recommended color palette for most purposes is Spectrum. This is not be confused with Rainbow, which is similar but is more heavily represented in the greens. Some other examples are seen below while omitting many options.